allostery ͉ chaperonin GroEL ͉ potassium ion ͉ timing mechanism M uch mechanistic and structural information related to the operation of the chaperonin nanomachine has accumulated (1-11). As with many other cellular machines, the chaperonin nanomachine has evolved to operate at variable speed in response to biological demand. This implies the presence of one or more timing devices or switches that govern the cycling of the machinery through the various functional and conformational states. In the presence of GroES, the two rings of GroEL operate 180°out of phase with one another in the manner of a two-stroke, reciprocating motor (1, 2). A complete chaperonin cycle consequently consists of two hemicycles and two reciprocal resting states (12).In the accompanying article (12), we used stopped-flow FRET experiments to monitor the association and dissociation of GroES to and from the asymmetric, resting-state complex comprising (GroEL cis -[ADP]-[GroES]/GroEL trans -ADP), where the square brackets indicate ligands that are not free to exchange with unbound ligand. We showed that the dominant timing mechanism is located on the trans ring. This timer is regulated by allosteric transitions in the trans ring that are both responsive to the nucleotides ADP and ATP, the potassium ion, and substrate protein (SP) and which are communicated to the cis ring. Here, our focus is on the hydrolysis of ATP. We show that the steady-state hydrolysis of ATP is largely governed by the trans ring timer and its response to the nucleotides ADP and ATP, K ϩ , and SP. We also show, paradoxically, that the hydrolysis of ATP in the newly formed cis ring is largely indifferent to the presence of encapsulated SP and to variations in [K ϩ ] that have profound effects on the rate of ATP hydrolysis in the steady state.

In a study of the timing mechanism of the chaperonin nanomachine we show that the hemicycle time (HCT) is determined by the mean residence time (MRT) of GroES on the cis ring of GroEL. In turn, this is governed by allosteric interactions within the trans ring of GroEL. Ligands that enhance the R (relaxed) state (residual ADP, the product of the previous hemicycle, and K ؉ ) extend the MRT and the HCT, whereas ligands that enhance the T (taut) state (unfolded substrate protein, SP) decrease the MRT and the HCT. In the absence of SP, the chaperonin machine idles in the resting state, but in the presence of SP it operates close to the speed limit, set by the rate of ATP hydrolysis by the cis ring. Thus, the conformational states of the trans ring largely control the speed of the complete chaperonin cycle.allostery ͉ GroEL ͉ timing mechanism ͉ GroES ͉ FRET P rotein machines and their man-made, macroscopic counterparts share several common attributes, e.g., concerted, coordinated movements, cyclical operation, and energy transduction. These machines are seldom reversible because each cycle generally involves at least one irreversible step, e.g., the consumption of fuel. Often these machines operate at variable speed, a plethora of timing devices adjusting the cycle speed in response to demand.An exemplary bipartite protein machine is the chaperonin system, typified by GroEL and GroES from Escherichia coli. GroEL is composed of 2 heptameric rings, stacked back to back, which, in the presence of GroES, operate out of phase with one another in the manner of a 2-stroke, reciprocating motor (1, 2). Driven by the hydrolysis of ATP, the chaperonin proteins function as a biological simulated annealing machine (3, 4), optimizing the folding of their substrate proteins (SPs) whose passage to biologically functional conformations is thus assured. A large body of literature dealing with many mechanistic and structural aspects has accumulated (for reviews, see refs. 5-11). However, despite this progress, surprisingly little is known about the timing mechanism of the chaperonin machine (however, see ref. 12). Here, we explore the location and operation of this timing device. We show that the timer is located on the trans ring and that it is regulated by allosteric transitions responsive to the nucleotides ADP and ATP, the potassium ion, and SPs.The subunits of the chaperonin machine display 2 forms of cooperativity: positive cooperativity between the subunits of 1 ring and negativity cooperativity between the subunits of different rings. This is well described by the nested model of allostery, extensively applied to the chaperonin system by Horovitz and colleagues (8). Briefly, ATP is bound and hydrolyzed preferentially, but not exclusively, to the relaxed (R) conformation. The influence of K ϩ is paradoxical. On one hand, it is needed for ATP hydrolysis in the steady state (13). However, at saturating concentrations of ATP, the rate of ATP hydrolysis is inversely proportional to the K ϩ concentration: the higher the K ϩ concentra...